Wt1-interacting protein WTIP

ABSTRACT

An isolated WT1 interacting protein having the amino acid sequence as set forth in SEQ ID NO: 2, or an isolated protein that has an amino acid sequence in which one or a plurality of amino acids have been substituted, deleted, inserted, and/or added is functionally equivalent to the protein having the amino acid sequence as set forth in SEQ ID NO: 2; and comprises the amino acid sequence from Glu at position  449  to Met at position  541  in SEQ ID NO: 2.

This application is a 371 of PCT/JP01/00461 filed on Jan. 24, 2001 andclaims benefit of JAPAN 2000-014949 filed on Jan. 24, 2000.

FIELD OF THE INVENTION

The present invention relates to a protein (WT1 interacting protein)(WTIP) that interacts with the WT1 protein, a gene encoding the same,and uses thereof.

BACKGROUND ART

WT1 (Wilms' tumor gene 1) gene is a transcription regulatory factor(Call K. M. et al., Cell 60, 509–520, 1990; Gessler M. et al., Nature343, 774–778, 1990) discovered in the course of identifying a causativegene of Wilms' tumor which is a pediatric kidney tumor, and functions asa tumor suppressor gene in at least some Wilms' tumors. It was alsodiscovered that its level of expression is high in most leukemia cells,and it is becoming clear that its level of expression at the firstmedical examination for leukemia correlates well with prognosis, and itis extremely useful as a marker of minimal residual disease (MRD) ofleukemia (Inoue K. et al., Blood 84, 3071–3079, 1994).

In normal tissue, WT1 is highly expressed in the testis, the ovary, thespleen, the mesenchymal mesothelium as well as in the embryonal kidney.Among malignant tumors, it is also highly expressed in leukemia,malignant mesothelioma, and solid tumors such as lung cancer. There isincreasing evidence that the WT1 gene is a gene having a variety offunctions in organogenesis, oncogenesis and the like (Reddy J. C. etal., Biochim. Biophys. Acta. 1287, 1–28, 1996; Davices R. et al., CancerRes. 59, 1747–1751, 1999).

The WT1 gene is mainly translated into four proteins by alternativesplicing in the exons (A in FIG. 1). The longest gene product, in whichexon 5 comprising 17 amino acid residues (17AA) and three amino acidresidues (KTS¹) in between the third and the fourth Zinc fingers havebeen inserted, is designated herein as WT1(+/+).

It has been demonstrated that the KTS-containing WT1(+/+) has a weak DNAbinding ability and binds to the mRNA splicing protein, whereas theWT1(+/−) having a potent DNA-binding ability functions as atranscription regulatory factor.

The WT1 protein is roughly composed of two regions, i.e., the functionregulatory region and the DNA-binding region containing zinc fingers. Inthe function regulatory region, as shown in A in FIG. 1, have twodomains that suppress or activate transcription. The assumption thatproteins that bind to these regions may be responsible for theregulation of functions led to the discovery of various WT1 interactingproteins. They include various proteins such as p53 (Malheswran S. etal., Proc. Natl. Acad. Sci. USA 90, 5100–5104, 1993), ubiquitineconjugating enzyme 9 (Wany Z. Y. et al., J. Biol. Chem. 271,24811–24816, 1996), par-4 (Johnstone R. W. et al., Mol. Cell Biol. 16,6945–6956, 1990), U2AF65 (Ravis R. C. et al., Geves. Rev. 12, 3217–3225,1998), and hsp70 (Maheswaran S. et al., Geves Rev. 12, 1108–1120, 1998).However, there are no reports of specific binding proteins in leukemiacells.

DISCLOSURE OF THE INVENTION

Thus, it is an object of the present invention to provide a novelprotein that interacts with (binds to) the WT1 protein, a gene encodingit, uses thereof and the like.

Thus, the present invention provides (1) a WT1 interacting proteinhaving the amino acid sequence as set forth in SEQ ID NO: 2, or aprotein that has an amino acid sequence in which one or a plurality ofamino acids have been substituted, deleted, inserted, and/or added inthe amino acids of said protein and that is functionally equivalent tothe protein having the amino acid sequence as set forth in SEQ ID NO: 2.

The present invention also provides (2) a protein that is encoded by DNAhybridizing under a stringent condition to DNA having the nucleotidesequence as set forth in SEQ ID NO: 1 and that is functionallyequivalent to the protein having the amino acid sequence as set forth inSEQ ID NO: 2.

The present invention also provides (3) a WT1 interacting proteincomprising an amino acid sequence from positions 449 to 541 in SEQ IDNO: 2.

The present invention also provides (4) a function modulator of the WT1protein comprising, as an active ingredient, a polypeptide comprising anamino acid sequence from Glu at position 449 to Met at position 541 inSEQ ID NO: 2.

The present invention also provides (5) a partial peptide of the proteindescribed in the above (1) or (2).

The present invention also provides (6) a gene encoding a proteindescribed in any of the above (1) to (3).

The present invention also provides (7) a vector comprising the genedescribed in the above (6).

The present invention also provides (8) a host cell carrying the vectordescribed in the above (7).

The present invention also provides (9) a method of producing a proteindescribed in any of the above (1) to (3), said method comprisingculturing the host cell of the above (8).

The present invention also provides (10) an antibody to a proteindescribed in any of the above (1) to (3).

The present invention also provides (11) a method of detecting ordetermining a protein described in any of the above (1) to (3)comprising:

(a) bringing the antibody described in the above (10) into contact witha sample expected to contain said protein; and

(b) detecting or determining the formation of an immune complex betweensaid antibody and said protein or immunostaining the cells that expresssaid protein using said antibody.

The present invention also provides (12) DNA that specificallyhybridizes to DNA comprising the base sequence as set forth in SEQ IDNO: 1 and that has a chain length of at least 15 bases.

The present invention also provides (13) a method of screening acompound that binds to a protein described in any of the above (1) to(3), said method comprising the steps of:

(a) bringing a sample to be tested into contact with said protein or apartial peptide thereof;

(b) detecting the binding activity of said sample with said protein or apartial peptide thereof; and

(c) selecting a compound having an activity of binding to said proteinor a partial peptide thereof.

The present invention also provides (14) a compound that can be isolatedby the method described in the above (13) and that binds to a proteindescribed in any of the above (1) to (3).

The present invention also provides (15) a method of screening acompound that promotes or inhibits the activity of a protein describedin any of the above (1) to (3), said method comprising the steps of:

(a) culturing the cells that express said protein in the presence of asample to be tested;

(b) detecting the growth of said cells; and

(c) selecting a compound that promotes or inhibits said growth ascompared to when detected in the absence of said sample to be tested.

The present invention also provides (16) a compound that can be isolatedby the method described in the above (15) and that promotes or inhibitsthe activity of a protein described in any of the above (1) to (3).

The present invention also provides (17) a method of screening acompound that promotes or inhibits the binding of the WT1 interactingprotein described in any of the above (1) to (3) with the WT1 protein,said method comprising the steps of:

(a) allowing the WT1 interacting protein to react with the WT1 proteinin the presence of a sample to be tested;

(b) determining the binding activity of both proteins; and

(c) selecting a compound that promotes or inhibits said binding ascompared to when detected in the absence of said sample to be tested.

The present invention also provides (18) a compound that can be isolatedby the method described in the above (17) and that promotes or inhibitsthe binding of the WT1 interacting protein described in any of the above(1) to (3) with the WT1 protein.

BRIEF EXPLANATION OF THE DRAWINGS

In FIG. 1, A is a drawing that shows the structure of cDNA encoding thefull-length of the WT1 protein, whereas B is a drawing that shows thestructure of a fusion protein (GST-WT2) of the suppressive domain of theWT1 protein and GST (glutathione S-transferase) and a fusion protein(GST-WT3) of the suppressive domain and the activating domain and GST,and a fusion protein (GST-WT4) of the activating domain and GST.

FIG. 2 is an electrophoretogram that shows the reactivity of theextracts of the K562 cells with the fusion protein shown in B of FIG. 1and with GST alone. It shows that there is an about 115 kDa proteinpresent in the extract of the K562 cells, that binds to the suppressivedomain of the WT1 protein.

FIG. 3 is a drawing that shows an elution profile wherein the cell-freeextracts (the centrifugation supernatant after the sonic disruption ofthe cells) of the K562 cells were separated by an anion exchangechromatography with HiLoad 16/10 Q Sepharose High Performance. Activitywas observed in fraction Nos. 26–35 by the west western blot method.

FIG. 4 is a drawing that shows an elution profile wherein the activefractions obtained in FIG. 3 were separated by an anion exchangechromatography with MONO Q HR 5/5. Activity was observed in fractionNos. 14 to 17 by the west western blot method.

FIG. 5 is a drawing that shows an elution profile wherein the activefractions obtained in FIG. 5 were separated by a hydrophobicchromatography with Phenyl Superrose HR 5/5. Activity was observed infraction Nos. 13 to 14 by the west western blot method.

FIG. 6 is an electrophoretogram that shows the result of proteindetection with Coomassie brilliant blue (CBB), and the west western blotmethod detection by the reactivity with GST-WT3, GST-WT2 and GST afterthe active fractions obtained in FIG. 5 containing 115 kDa protein weretransferred to a PVDF membrane. It can be seen that the 115 kDa proteinbinds to the suppressive domain of WT1.

In FIG. 7, A shows the structure of the full-length WTIP, B shows thestructure of polypeptides (WTIP 1-406 and WTIP 1-178) containing the WWdomain at the N-terminal end of WTIP and a polypeptide (WTIP 1-98)containing no WW domains in which a histidine (His) tag (tag) has beenattached to the C-terminal of each polypeptide.

FIG. 8 is an electrophoretogram that shows the result of detection bythe reactivity with anti-His tagged polypeptide antibody, GST-WT3, andGST after the polypeptide shown in FIG. 7 was electrophoresed. It can beseen that WTIP 1-406 and WTIP 1-178 containing the WW domain react, butthat WTIP 1-98, that does not contain the WW domain, does not react,with GST-WT3 that contains the suppressive domain, and GST that does notcontain the suppressive domain does not react with any of the WTIPfragments, indicating that the WW domain in WTIP and a part of thesuppressive domain of the WT1 protein react.

FIG. 9 is an electrophoretogram that shows that WT1 and WTIP bind in thecell.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

In order to obtain a novel protein that binds to the WT1 protein, WT1interacting proteins were searched for using a human leukemia cell lineK562 (Yamagami T. et al., Blood 87,2878–2885, 1996) in which growthinhibition is observed when using an antisense oligo DNA of WT1. In awest western blot using a fusion protein of the WT1 regulatory regionand glutathione S-transferase (GST), an intracellular protein that bindsto the fusion protein was noted, which thereby was separated by a columnchromatography and SDS-PAGE, and a gene was identified by peptidemapping and by screening with expression. As a result, cDNA of a novelgene WT1 interacting protein (WTIP) was cloned.

Thus the present invention provides a WT1 interacting protein having theamino acid sequence as set forth in SEQ ID NO: 1.

In the sequence described in SEQ ID NO: 1, the nucleotide sequence ofpositions 1 to 1358 corresponds to FBP11 (Chan D. C. et al., EMBO J. 15,1045–1054, 1996) which is one of the proteins (formin binding protein)binding to formin involved in the development of mouse legs and kidneyand HYPA (Huntington yeast partner A) (Faber P. W. et al., Hum. Mol.Genet. 7, 1463–1467, 1998) which is one of the proteins binding toHuntington that is considered to be a causative gene of Huntington'schorea, a human neurodegenerative disease, and the amino acid sequencecorresponding to the nucleotide sequence of positions 1636 to 2980 inSEQ ID NO: 1 corresponds to NY-REN-6 (Scanlan M. J. et al., Int. J.Cancer 83, 456–464, 1999) identified as an autoantigen that recognizesan autoantibody in the serum of human patients with kidney cancer.

Thus, a protein having a full-length and partial amino acid sequence,comprising the amino acid sequence corresponding to the nucleotidesequence of positions 1359 to 1635 in SEQ ID NO: 1, is novel. Thus, thepresent invention provides a protein comprising an amino acid sequencethat corresponds to the nucleotide sequence of positions 1359 to 1635 inSEQ ID NO: 1.

The present invention also provides a gene, for example DNA or RNA,encoding the above protein. Said DNA is specifically cDNA.

The present invention also provides a vector comprising the above DNA,for example an expression vector.

In accordance with the present invention, it was found that the WWdomain from Gly at position 141 to Asp at position 217 in SEQ ID NO: 2is a binding region with the WT1 protein. Thus the present inventionprovides a regulatory agent of WT1 protein function comprising, as anactive ingredient, a polypeptide comprising an amino acid sequence fromGly at position 141 to Asp at position 217 in SEQ ID NO: 2. Inaccordance with the present invention, a polypeptide having an aminoacid sequence of position 1 (Met) to position 98 (Gly) in SEQ ID NO: 2does not bind to the WT1 protein, but a polypeptide having an amino acidsequence of position 1 (Met) to position 178 (Ala) binds to the WT1protein. Thus, according to one aspect of the present invention, thereis provided a regulatory agent of WT1 protein function comprising as anactive ingredient a polypeptide having or comprising an amino acidsequence from position 99 (Gln) to position 178 (Ala) in SEQ ID NO: 2.

In accordance with the present invention, a polypeptide having an aminoacid sequence of position 1 (Met) to position 406 (Glu) in SEQ ID NO: 2binds to the WT1 protein. Thus, according to one aspect of the presentinvention, there is provided a regulatory agent of WT1 protein functioncomprising, as an active ingredient, a polypeptide having or comprisingan amino acid sequence from position 99 (Gln) to position 406 (Glu) inSEQ ID NO: 2. More generally, the present invention provides aregulatory agent of WT1 protein function comprising, as an activeingredient, a polypeptide or a protein from any amino acid of position 1(Met) to position 99 (Gln) to any amino acid of position 189 (Glu) toposition 406 (Glu) or position 957 (Gin) in SEQ ID NO: 2.

The isolation of WTIP and the cloning of cDNA encoding it is describedin Example 1. In the example, human leukemia cell-like K562 (JapaneseCancer Research Resource Banks) was used as a source for isolating WTIPand DNA encoding it, but other cells that produce WTIP may also be used.

The present invention encompasses proteins functionally equivalent tothe WT1 interacting protein. Such proteins include homolog proteins ofother organisms corresponding to the human WT1 interacting protein andmutants of the human WT1 interacting protein. As used herein the term“functionally equivalent” means that the subject protein has abiological activity equivalent to the above WT1 interacting protein. Thebiological activity is, for example, a binding activity with the WT1protein.

As a method well known to a person skilled in the art for preparing aprotein functionally equivalent to a certain protein, there is known amethod of introducing mutation in the protein. For example, a personskilled in the art can use site-directed mutagenesis (Hashimoto-Gotoh,T. et al. (1995) Gene 152, 271–275; Zoller, M. J. and Smith, M. (1983)Methods Enzymol. 100, 468–500; Kramer, W. et al. (1984) Nucleic AcidsRes. 12, 9441–9456; Kramer W. and Fritz H. J. (1987) Methods Enzymol.154, 350–367; Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82,488–492; Kunkel (1988) Methods Enzymol. 85, 2763–2766) to introduce asappropriate a mutation in amino acids of the human WT1 interactingprotein in order to prepare a protein functionally equivalent to thehuman WT1 interacting protein.

Mutation in amino acids also occurs spontaneously in nature. Thus,proteins that have an amino acid sequence in which one or a plurality ofamino acids are mutated in the amino acid sequence of the human WT1interacting protein and that are functionally equivalent the human WT1interacting protein are also encompassed in the present invention.

As proteins functionally equivalent to the WT1 interacting protein ofthe present invention, there can be specifically mentioned those inwhich one or more than one, preferably two or more and 30 or less, morepreferably two or more and 10 or less amino acids have been deleted inthe amino acid sequence as set forth in SEQ ID NO: 2, those in which oneor more than one, preferably two or more and 30 or less, more preferablytwo or more and 10 or less amino acids have been added in the amino acidsequence as set forth in SEQ ID NO: 2, and those in which one or morethan one, preferably two or more and 30 or less, more preferably two ormore and 10 or less amino acids have been substituted with other aminoacids in the amino acid sequence as set forth in SEQ ID NO: 2.

For the amino acid residue that is mutated, it is preferably mutated toanother amino acid that retains the property of the amino acid sidechain. For example, as the properties of amino acid side chains, theremay be mentioned hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acidshaving an aliphatic side chain (G, A, V, L, I, P), amino acids having ahydroxy group-containing side chain (S, T, Y), amino acids having asulfur-containing side chain (C, M), amino acids having a carboxylicacid- or amide-containing side chain (D, N, E, Q), amino acids having abase-containing side chain (R, K, H), and amino acids having anaromatic-containing side chain (H, F, Y, W) (all letters in parenthesesindicate a one-letter representation of amino acids).

It is already known that proteins having an amino acid sequence that hasbeen modified by deletion, addition of one or a plurality of amino acidresidues and/or substitution with another amino acid in a certain aminoacid sequence can retain the biological activity (Mark, D. F. et al.,Proc. Natl. Acad. Sci. USA (1984) 81, 5662–5666; Zoller, M. J. & Smith,M., Nucleic Acids Research (1982) 10, 6487–6500; Wang, A. et al.,Science 224, 1431–1433; Dalbadie-McFarland, G. et al., Proc. Natl. Acad.Sci. USA (1982) 79, 6409–6413).

As a protein in which one or a plurality of amino acids have been addedto the amino acid sequence (SEQ ID NO: 2) of the human WT1 interactingprotein, there can be mentioned a fusion protein that contains the humanWT1 interacting protein. A fusion protein is a fusion of the human WT1interacting protein and another peptide or protein, and is included inthe present invention. In a method of creating a fusion protein, DNAencoding the human WT1 interacting protein of the present invention andDNA encoding another peptide or protein are linked in a frame with eachother, which is then introduced into an expression vector and expressedin a host cell, and a method well known to a person skilled in the artcan be used. Any other peptide or protein that is subjected to fusionwith the protein of the present invention is not specifically limited.

As another peptide that is subjected to fusion with the protein of thepresent invention, for example, known peptides may be used includingFLAG (Hopp, T. P. et al., BioTechnology (1988) 6, 1204–1210) , 6×His(SEQ ID NO: 13) comprising 6 His (histidine) (SEQ ID NO: 13) residues,10×His (SEQ ID NO: 14), influenza hemagglutinin (HA), fragments of humanc-myc, fragments of VSV-GP, fragments of p18HIV, T7-tag, HSV-tag, E-tag,fragments of SV40T antigen, lck tag, fragments of α-tubulin, B-tag,fragments of Protein C, and the like. As another protein that issubjected to fusion with the protein of the present invention, there maybe mentioned, for example, GST (glutathione S-transferase), HA(influenza agglutinin), the constant regions of immunoglobulin,β-galactosidase, MBP (maltose-binding protein), and the like.

DNA encoding such a peptide or protein is fused to DNA encoding theprotein of the present invention, and the fused DNA thus prepared isexpressed to prepare the fusion protein.

As a method well known to a person skilled in the art for preparing aprotein functionally equivalent to a certain protein, there can bementioned a method that employs the hybridization technology (Sambrook,J. et al., Molecular Cloning 2nd ed., 9.47–9.58, Cold Spring HarborLaboratory Press, 1989).

Thus, it can be routinely performed by a person skilled in the art thata DNA sequence (SEQ ID NO: 1) encoding the human WT1 interacting proteinor a portion thereof is used to isolate DNA having a high homologytherewith, and from said DNA a protein functionally equivalent to thehuman WT1 interacting protein can be routinely isolated. Thus, a proteinencoded by DNA that hybridizes to DNA encoding the human WT1 interactingprotein or DNA comprising a portion thereof and that is functionallyequivalent to the human WT1 interacting protein is also included in thepresent invention.

As such a protein, there can be mentioned a homolog from a mammal otherthan the human (for example, a protein encoded by a monkey, mouse, rat,rabbit, and bovine gene). When a cDNA having a high homology with DNAencoding the human WT1 interacting protein is isolated from an animal,preferably tissues such as the heart, the placenta, the testis and thelike are used.

A hybridization condition for isolating a DNA encoding a proteinfunctionally equivalent to the human WT1 interacting protein may beselected, as appropriate, by a person skilled in the art. As thehybridization condition, there can be mentioned a low stringentcondition. A low stringent condition is, for example, 42° C., 2×SSC, and0.1% SDS, and preferably 50° C., 2×SSC, and 0.1% SDS.

More preferably, there can be mentioned a high stringent condition. As ahigh stringent condition, for example, there can be mentioned 65° C.,2×SSC, and 0.1% SDS. In these conditions, the higher the temperature isthe higher the homology of the DNA obtained is. However, as elementsaffecting the stringency of hybridization, a plurality of elements canbe conceived such as a salt concentration in addition to temperature,and a person skilled in the art can select, as appropriate, theseelements to attain a similar stringency.

Isolation can also be attained by utilizing a gene amplifying methodusing primers synthesized based on the sequence information of DNA (SEQID NO: 1) encoding the human WT1 interacting protein, for example apolymerase chain reaction (PCR), in place of hybridization.

A protein functionally equivalent to the human WT1 interacting proteinencoded by the DNA isolated by such a hybridization technology or geneamplification technology usually has a high homology with the human WT1interacting protein in terms of the amino acid sequence.

The protein of the present invention also includes a protein that isfunctionally equivalent to the human WT1 interacting protein and thathas a high homology with the amino acid sequence as set forth in SEQ IDNO: 2. A high homology as used herein means a homology of 70% or higher,preferably 80% or higher, more preferably 90% or higher, and morepreferably 95% or higher in identity. In order to determine the homologyof proteins, an algorithm described in an article (Wilbur, W. J. andLipman, D. J., Proc. Natl. Acad. Sci. USA (1983) 80, 726–730) may beused.

The protein of the present invention may differ in amino acid sequence,molecular weight, isoelectric point, or the presence or absence andshapes of sugar chains depending on the cells, described below, thatproduce it, the host cells, and the method of purification. However, itis included in the present invention as long as the protein obtainedretains functions equivalent to the human WT1 interacting protein of thepresent invention (SEQ ID NO: 2). For example, when the protein of thepresent invention is expressed in prokaryotic cells such as Escherichiacoli (E. coli), a methionine residue is added to the N-terminal of theamino acid sequence of the original protein. Also, when it is expressedin eukaryotic cells such as mammalian cells, the signal sequence at theN-terminal is removed. The protein of the present invention encompassessuch proteins.

The protein of the present invention can be prepared by a person skilledin the art in a known method either recombinantly or as natural protein.In the case of recombinant proteins, DNA (such as DNA having the basesequence as set forth in SEQ ID NO: 1) encoding a protein of the presentinvention is integrated into a suitable expression vector, which in turnis introduced into a suitable host cell and a recombinant is recoveredtherefrom to obtain an extract. Then the extract is purified bysubjecting it to ion exchange, reverse-phase, gel filtrationchromatography, or affinity chromatography in which an antibody againstthe protein of the present invention has been immobilized on the column,or by using a combination of a plurality of columns to prepare theprotein of the present invention.

When the protein of the present invention was expressed as a fusionprotein with the glutathione S-transferase protein or as a recombinantprotein to which a plurality of histidines were added in a host cell(for example, in an animal cell or E. coli), the expressed recombinantprotein can be purified by a glutathione column or a nickel column.

After purification of the fusion protein, the regions other than theprotein of interest among the fusion protein may be removed, as desired,by cleaving with thrombin, Factor Xa etc.

In the case of a natural protein, it can be purified and isolated bysubjecting an extract of a tissue or cell that expresses the protein ofthe present invention it to an affinity column to which an antibody,described below, that binds to the WT1 interacting protein is bound. Theantibody may be a polyclonal antibody or a monoclonal antibody.

The present invention also encompasses the partial peptides of theprotein of the present invention. The partial peptides comprising theamino acid sequence specific to the protein of the present inventioncomprises at least 7 amino acids, preferably 8 amino acids or more, andmore preferably 9 amino acids or more. Said partial peptides can be usedfor preparing an antibody against the protein of the present invention,screening compounds that bind to the protein of the present invention,and screening promoters and inhibitors of the protein of the presentinvention. Furthermore, they can be an antagonist to ligands of theprotein of the present invention.

As the partial peptides of the present invention, for example, there canbe mentioned partial peptides comprising an active center of the proteincomprising the amino acid sequence as set forth in SEQ ID NO: 2. Therecan also be mentioned partial peptides containing one or a plurality ofregions of the hydrophobic regions and hydrophilic regions deduced fromthe hydrophobicity plot analysis. These partial peptides may containpart or all of a hydrophobic region, or contain part or all of ahydrophilic region.

The partial peptide of the present invention can be produced by geneengineering technology, known peptide synthetic methods, or by cleavingthe protein of the present invention with a suitable peptidase. As thepeptide synthesis method, any of a solid-phase synthesis and aliquid-phase synthesis can be used.

The present invention also relates to DNA encoding the protein of thepresent invention. The DNA of the present invention can be used for thein vivo or in vitro production of the protein of the present inventionand, besides, the application in gene therapy to diseases associatedwith aberrations in the gene encoding the protein of the presentinvention is contemplated.

The DNA of the present invention may be in any form as long as it canencode the protein of the present invention. Thus, it can be any of cDNAsynthesized from mRNA, genomic DNA, or chemically synthesized DNA.Furthermore, it contains DNA having any base sequence based on thedegeneracy of genetic code, as long as it encodes the protein of thepresent invention.

The DNA of the present invention can be prepared by any method known toa person skilled in the art. For example, it can be prepared bypreparing a cDNA library from the cells expressing the protein of thepresent invention and subjecting it to hybridization using, as theprobe, part of the DNA sequence (for example SEQ ID NO: 1) of thepresent invention. cDNA libraries may be prepared by a method describedin Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor LaboratoryPress (1989), or commercially available DNA libraries may be used. It isalso possible to prepare RNA from the cells expressing the protein ofthe present invention, synthesizing an oligo DNA based on the DNAsequence (for example SEQ ID NO: 1) of the present invention, which isthen used as a primer in a PCR reaction to amplify cDNA encoding theprotein of the present invention.

By determining the nucleotide sequence of the cDNA obtained, thetranslation region encoding it can be determined, and thereby the aminoacid sequence of the protein of the present invention can be obtained.Furthermore, genomic DNA can be isolated by screening a genomic DNAlibrary using the obtained cDNA as the probe.

Specifically the following method can be followed. First, mRNA isisolated from cells, tissues, or organs (for example, the ovary, thetestis, the placenta etc.) expressing the protein of the presentinvention. The isolation of mRNA can be effected by preparing total RNAusing a known method such as the guanidine ultracentrifugation method(Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294–5299), the AGPCmethod (Chomczynski, P. and Sacchi, N., Anal. Biochem. (1987) 162,156–159) and the like, and then by purifying mRNA from total RNA usingthe mRNA Purification Kit (Pharmacia) and the like. mRNA can also beprepared directly by using the QuickPrep mRNA Purification Kit(Pharmacia).

cDNA is synthesized from the mRNA obtained using a reversetranscriptase. The synthesis of cDNA can be effected using the AMVReverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Kogyo),and the like. Alternatively, for the synthesis and amplification ofcDNA, the 5′-Ampli FINDER RACE kit (manufactured by Clontech) and the5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. U.S.A.(1988) 85, 8998–9002; Belyavsky, A. et al., Nucleic Acids Res. (1989)17, 2919–2932), that employs the polymerase chain reaction (PCR), may beused.

A DNA fragment of interest may be prepared from the PCR product thusobtained and ligated to a vector DNA. Furthermore, a recombinant vectoris constructed from this, which is then introduced into E. coli forselection of colonies to prepare the desired recombinant vector. Thebase sequence of the desired DNA may be confirmed by a known method suchas the dideoxy nucleotide chain termination method.

Considering the frequency of use of the host's codon for use in thepresent invention, a sequence having a better efficiency of expressioncan be designed (Grantham, R. et al., Nucleic Acids Research (1981) 9,r43–r74). Furthermore, the DNA of the present invention may be alteredby using commercially available kits or by known methods. Alternativeincludes, for example, digestion with a restriction enzyme, insertion ofa synthetic oligonucleotide or a suitable DNA fragment, addition of alinker, insertion of an initiation codon (ATG) and/or a stop codon (ATT,TGA or TAG), and the like.

The DNA of the present invention includes DNA that hybridizes to the DNAcomprising the base sequence as set forth in SEQ ID NO: 1 under astringent condition and DNA that encodes a protein functionallyequivalent to the above protein of the present invention.

As the stringent condition, which can be chosen as appropriate by aperson skilled in the art, there can be mentioned a low stringentcondition. A low stringent condition is, for example, 42° C., 2×SSC, and0.1% SDS, and preferably 50° C., 2×SSC, and 0.1% SDS. More preferably,there can be mentioned a high stringent condition. As a high stringentcondition, for example, there can be mentioned 65° C., 2×SSC, and 0.1%SDS. In these conditions, the higher the temperature is the higher thehomology of DNA obtained is. The above hybridizing DNA may preferably benaturally occurring DNA, for example cDNA or chromosomal DNA.

The present invention also relates to a vector in which the DNA of thepresent invention has been inserted. The vector of the present inventionis useful in retaining the DNA of the present invention in the host celland in expressing the protein of the present invention.

The vector is not specifically limited, as long as it has “ori” for usein amplification in E. coli to produce and amplify in large quantitiesthe vector in E. coli (for example, JM109, DH5α, HB101, XL1Blue) when E.coli is used as the host, and a selection gene (for example a drugresistance gene that can be identified by a drug such as ampicillin,tetracycline, kanamycin, and chloramphenicol) in the transformed E.coli.

Examples of vectors include M13 vectors, pUC vectors, pBR322,pBluescript, pCR-Script and the like. Also, for the purpose ofsubcloning and excising of DNA, pGEM-T, pDIRECT, pT7 etc., in additionto the above vector, may be mentioned. When a vector is used to producethe protein of the present invention specifically, the use of anexpression vector is useful.

When expression is to be effected in E. coli, the expression vectorshould have the above characteristics so that it can be expressed in E.coli, and besides, when the host is such E. coli as JM109, DH5α, HB101,XL1-Blue etc., it must have a promoter that permits efficient expressionin E. coli, such as the lacz promoter (Ward et al., Nature (1989) 341:544–546; FASEB J. (1992) 6, 2422–2427), the ara B promoter (Better etal., Science (1988) 240, 1041–1043), the T7 promoter, or the like. Assuch vectors, there may be mentioned, in addition to the above vectors,pGEX-5X-1 (manufactured by Pharmacia), “QIAexpress system” (manufacturedby Qiagen), pEGFP, pET (in this case, the host is preferably BL21 thatis expressing T7 RNA polymerase), or the like.

The vector can also contain a signal sequence for polypeptide secretion.As the signal sequence for protein secretion, when produced in theperiplasm of E. coli, the pelB signal sequence (Lei, S. P. et al., J.Bacteriol. (1987) 169, 4379) can be used. The introduction of a vectorinto the host cell may be effected by, for example, the calcium chloridemethod and the electroporation method.

For the production of the protein of the present invention, in additionto E. coli, there can be mentioned expression vectors derived frommammals (for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS(Nucleic Acids Res. 18 (17), 5322, 1990), pEF, pCDM8), expressionvectors derived from insect cells (for example, “Bac-to-Bac baculovirusexpression system” (manufactured by GIBCO BRL), pBacPAK8), expressionvectors derived from plants (for example, pMH1, pMH2), expressionvectors derived from animal viruses (for example, pHSV, pMV, pAdexLcw),expression vectors derived from retrovirus vectors (for example,pZIpneo), expression vectors derived from yeast (for example, “PichiaExpression Kit” (manufactured by Invitrogen), pNV11, SP-Q01), expressionvectors derived from Bacillus subtilis (for example, pPL608, pKTH50),and the like.

For the purpose of expressing in animal cells such as CHO cells, COScells, NIH3T3 cells and the like, it is indispensable for the vectors tohave promoters required for expression in cells such as SV40 promoter(Mulligan et al., Nature (1979) 277, 108), MMLV-LTR promoter, EF1αpromoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMVpromoter and the like, and more preferably to have genes (such as a drugresistant gene that can be identified by a drug such as neomycin, G418)for selecting transformation into the cell. As vectors having suchcharacteristics, there can be mentioned for example PMAM, pDR2, PBK-RSV,PBK-CMV, pOPRSV, pOP13 and the like.

Furthermore, for the purpose of stably expressing a gene and amplifyingthe copy number of a gene in the cell, there may be mentioned a methodin which a vector (for example, pCHOI) having the DHFR gene isintroduced into the CHO cells deficient in the nucleic acid syntheticpathway to complement the deficiency and is amplified with methotrexate(MTX). For the purpose of transient expression of a gene, there may bementioned a method in which transformation is effected with a vector(pcD etc.) having the origin of replication for SV40 using COS cellshaving on the chromosome a gene that expresses the SV40 T antigen.

As the origin of replication, there can be used those derived frompolyoma virus, adenovirus, bovine papilloma virus (BPV) and the like.Furthermore, for the amplification of gene copy numbers in the host cellsystem, expression vectors can include, as selectable markers, theaminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene,E. coli xanthine guaninephosphoribosyl transferase (Ecogpt) gene, thedihydrofolate reductase (dhfr) gene and the like.

On the other hand, as methods of expressing the DNA of the presentinvention in an organism, there can be mentioned a method in which theDNA of the present invention is integrated into a suitable vector, andthen introduced into an organism by the retrovirus method, the liposomemethod, the cationic liposome method, the adenovirus method and thelike. This permits the gene therapy of diseases associated with mutationin the gene of the present invention. As the vector used, there can bementioned, but not limited to, an adenovirus vector (for example,pAdexlcw), a retrovirus vector (for example, pZIPneo) and the like.Common genetic manipulation such as the insertion of the DNA of thepresent invention into a vector may be performed by a standard method(Molecular Cloning, 5.61–5.63). The administration into an organism maybe an ex vivo method or an in vivo method.

The present invention also relates to a host cell into which the vectorof the present invention has been introduced. The host cell into whichthe vector of the present invention is introduced is not specificallylimited, and E. coli and various animal cells may be used. For example,the host cell of the present invention can be used for the production ofthe protein of the present invention or as a production system forexpression. The production system of protein preparation comprises thein vitro and the in vivo production system. As an in vitro productionsystem, there can be mentioned a production system which employseukaryotic cells and a production system which employs prokaryoticcells.

When the eukaryotic cells are used, there are the production systemswhich employ the animal cells, the plant cells, and the fungal cells.Known animal cells include mammalian cells such as CHO cells (J. Exp.Med. (1995) 108, 945), COS cells, 3T3 cells, myeloma cells, baby hamsterkidney (BHK) cells, HeLa cells and Vero cells, amphibian cells such asXenopus oocytes (Valle, et al., Nature (1981) 291, 358–340), or insectcells such as sf9, sf21, and Tn5.

As the CHO cells, dhfr-CHO (Proc. Natl. Acad. Sci. U.S.A. (1968) 77,4216–4220), a CHO cell that is deficient in the DHFR gene, and CHO K-1(Proc. Natl. Acad. Sci. U.S.A. (1968) 60, 1275) can be preferably used.In animal cells, for the purpose of large scale production, CHO cellsare specifically preferred. The introduction of a vector into the hostcell may be carried out by, for example, the calcium phosphate method,the DEAE-dextran method, the cationic ribozome DOTAP (manufactured byBoehringer Mannheim), the electroporation method, and the lipofectionmethod.

As plant cells, for example, there are known cells derived fromNicotiana tabacum as the protein production system, which may subjectedto callus culture. Known fungal cells include yeasts such as the genusSaccharomyces, more specifically Saccharomyces cereviceae, orfilamentous fungi such as the genus Aspergillus, more specificallyAspergillus niger.

When the prokaryotic cells are used, there are the production systemswhich employ bacterial cells. Known bacterial cells include Escherichiacoli (E. coli) such as JM109, DH5α and HB101, and Bacillus subtilis.

By transforming these cells with the desired DNA and culturing thetransformed cells in vitro, proteins can be obtained. Culturing isconducted using known methods. For example, as the culture liquid, DMEM,MEM, RPMI1640, and IMDM can be used, and hereupon serum supplements suchas fetal calf serum (FCS) may be used in combination, or serum-freemedium can be used. pH during the culture is preferably about 6 to 8.Culture is usually carried out at about 30 to 40° C. for about 15 to 200hours with, as desired, medium change, aeration, and agitation.

On the other hand, as in vivo production systems, there can be mentionedthose which employ animals and those which employ plants. The DNA ofinterest is introduced into these animals or plants, and the proteinsare produced in the body of such animals or plants, and recovered. Theterm “host cell” as used herein encompasses these animals and plants.

When animals are used, there are the production systems which employmammals and insects. As mammals, goats, pigs, sheep, mice, and cattlecan be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993).When mammals are used, transgenic animals can be used.

For example, the DNA of interest is prepared as a fusion gene with agene encoding protein which is inherently produced in the milk such asgoat β casein. The DNA fragment containing the fusion gene into whichthe DNA has been inserted is injected into a goat embryo, and the embryois introduced into a female goat. The protein of interest can beobtained from the milk produced by the transgenic goat borne to the goatwho received the embryo or the offspring thereof. In order to increasethe amount of milk containing the protein produced by the transgenicgoat, hormones may be given to the transgenic goat as appropriate.(Ebert, K. M. et al., Bio/Technology (1994) 12, 699–702).

As an insect, silkworms may be used. When silkworms are used,baculovirus into which the DNA of interest has been inserted is infectedto the silkworm, and the desired protein can be obtained from the bodyfluid of the silkworm (Susumu, M. et al., Nature (1985) 315, 592–594).

Moreover, when plants are used, tabacco, for example, can be used. Whentabacco is used, the DNA of interest is inserted into an expressionvector for plants, for example pMON 530, and then the vector isintroduced into a bacterium such as Agrobacterium tumefaciens. Thebacterium is then infected to tabacco such as Nicotiana tabacum toobtain the desired protein from the leaves of the tabacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131–138)

Proteins of the present invention thus obtained can be separated fromthe inside or outside (culture medium etc.) of the host cell and thenmay be purified as virtually pure and homogeneous proteins. Separationand purification of the antibody for use in the present invention may beaccomplished by, but not limited to, separation and the purificationmethods conventionally used for protein purification. Proteins can beseparated and purified by selecting and combining, as appropriate,methods including chromatography columns, filtration, ultrafiltration,salting-out, solvent precipitation, distillation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis,recrystallization and the like.

As chromatography, there may be mentioned, for example, affinitychromatography, ion exchange chromatography, hydrophobic chromatography,gel-filtration, reverse phase chromatography, adsorption chromatography,and the like (Strategies for Protein Purification and Characterization:A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press, 1996). These chromatographies can be carriedout using a liquid chromatography such as HPLC and FPLC. The presentinvention encompasses proteins that were highly purified using thesepurification methods.

Furthermore, before or after purification, proteins can be modified orpeptides can be partially removed as appropriate by acting a suitableprotein modifying enzyme. As the protein modifying enzyme, there can beused, for example, trypsin, chymotrypsin, lysyl endopeptidase, proteinkinase, and glucosidase.

The present invention also relates to antibody that binds to the proteinof the present invention. The form of the protein of the presentinvention is not specifically limited, and includes polyclonal antibodyas well as monoclonal antibody. Furthermore, antiserum obtained byimmunizing an immune animal such as a rabbit with the protein of thepresent invention, all classes of polyclonal antibodies and monoclonalantibodies, and moreover human antibodies and humanized antibodies bygene recombinant technology.

Though the protein of the present invention for use as the sensitizingantigen for generation of antibodies is not limited by the animalspecies from which the antibodies are obtained, it is preferably derivedfrom mammals such as humans, mice, or rats with the human-derivedprotein being most preferred. Human-derived protein can be obtainedusing the gene sequence or the amino acid sequence disclosed herein.

Proteins that are used as the sensitizing antigen herein may be completeproteins or partial peptides thereof. As the partial peptides of theproteins, for example, N-terminal fragments (N) or C-terminal fragments(C) of the proteins may be mentioned. The term “antibody” as used hereinmeans antibody that reacts to the full-length protein or fragmentsthereof.

A gene encoding the protein of the present invention or a fragmentthereof may be inserted into a known expression system, and said vectoris used to transform the host cell described herein to obtain theprotein of interest or a fragment thereof from the inside or outside ofsaid host cell by a known method, which may be used as the sensitizingantigen. Alternatively, the cell that expresses the protein or lysatesthereof or the chemically synthesized protein of the present inventionmay be used as sensitizing antigen.

Mammals to be immunized with the sensitizing antigen are notspecifically limited, and they are preferably selected in considerationof their compatibility with the parent cell for use in cell fusion. Theygenerally include animals of the order Rodentia, the order Lagomorpha,and the order Primates.

Animals of order.Rodentia include, for example, mice, rats, hamsters,and the like. Animals of order Lagomorpha include, for example, rabbits.Animals of order Primates include, for example, monkeys. As monkeys,catarrhines (Old-World monkeys) such as cynomolgi (crab-eating macaque),rhesus monkeys, sacred baboons, chimpanzees etc. are used.

Immunization of animals with a sensitizing antigen is carried out usinga known method. A general method, for example, involves theintraperitoneal or subcutaneous injection of a sensitizing antigen tothe mammal. Specifically, a sensitizing antigen which has been dilutedand suspended in an appropriate amount of phosphate buffered saline(PBS) or physiological saline is mixed, as desired, with an appropriateamount of a common adjuvant, for example Freund's complete adjuvant.After being emulsified, it is administered to the mammal. Then thesensitizing antigen dissolved at a suitable amount is Freund'sincomplete adjuvant is administered for several times every 4 to 21days. Alternatively a suitable carrier may be used at the time ofimmunization of the sensitizing antigen. After such immunization, theincrease in the desired antibody levels in the serum is confirmed by aconventional method.

In order to obtain polyclonal antibodies, the blood of the mammal thatwas sensitized with the antigen is removed after the increase in thedesired antibody levels in the serum has been confirmed. Serum isseparated from the blood by a known method. As polyclonal antibodies,serum containing the polyclonal antibodies may be used, or, as desired,the fraction containing the polyclonal antibodies may be isolated fromthe serum and used. For example, using an affinity column to which theprotein of the present invention has been coupled, a fraction thatrecognizes the protein of the present invention only is obtained, andthis fragment is purified using a Protein A or a Protein G column, toprepare an immunoglobulin G or M.

In order to obtain monoclonal antibodies, immune cells of the mammalthat was sensitized with the above antigen are removed and are subjectedto cell fusion after the increase in the desired antibody levels in theserum has been confirmed. At this time preferred immune cells that aresubjected to cell fusion include, in particular, the spleen cells. Themammalian myeloma cells as the other parent cells which are subjected tocell fusion with the above-mentioned immune cells preferably includemyeloma cells of a mammal, more preferably myeloma cells that haveacquired a characteristic feature for the selection of fusion cells by adrug.

Cell fusion between the above immune cells and the myeloma cells may becarried out according to the conventional method, for example, themethod of Milstein (Galfre, G. and Milstein. C, Methods Enzymol. (1981)73, 3–46) and the like.

The hybridoma obtained by cell fusion may be selected by culturing in aHAT culture medium (a culture liquid containing hypoxanthine,aminopterin, and thymidine). Culturing in said HAT culture medium iscontinued generally for a period of time sufficient to effect killing ofthe cells other than the desired hybridoma (non-fusion cells), generallyfor several days to several weeks. Then, the conventional limitingdilution method is conducted in which the hybridomas that produce thedesired antibody are screened and cloned.

In addition to obtaining the above hybridoma by immunizing an animalother than the human with an antigen, it is also possible to sensitizehuman lymphocytes, for example lymphocytes infected with EB virus, witha protein, cells expressing them or their lysates in vitro, and to allowthe resulting sensitized lymphocytes to be fused with a human-derivedmyeloma cell having a permanent division potential, for example U266,and thereby to obtain a hybridoma that produces the desired humanantibody having the activity of binding the protein (see JapaneseUnexamined Patent Publication (Kokai) No. 63-17688).

Then, the hybridoma obtained is transplanted into the abdominal cavityof a mouse, ascites is recovered from the mouse, and the monoclonalantibody obtained is purified and prepared by subjecting it to ammoniumsulfate precipitation, Protein A or Protein G column, DEAE ion exchangechromatography, affinity column to which the protein of the presentinvention has been coupled, and the like. The antibody of the presentinvention can be used for the purification and detection of the proteinof the present invention and, besides, becomes a candidate for theagonist and the antagonist of the protein of the present invention. Theantibody may be used for antibody therapy of diseases with which theprotein of the present invention is associated. When the antibodyobtained is used for the purpose (antibody therapy) of administering toa human body, it is preferably human antibody or humanized antibody inorder to reduce immunogenicity.

Furthermore, a transgenic animal having a repertoire of human antibodygenes is immunized with the antigen protein, cells expressing them orlysates thereof to obtain the antibody-producing cells, which are usedto obtain human antibody against the protein for use in the presentinvention using hybridomas fused to myeloma cells (see InternationalPatent Application WO 92-03918, WO 93-2227, WO 94-02602, WO 94-25585, WO96-33735 and WO 96-34096).

In addition to using a hybridoma to produce antibody, antibody-producingimmune cells such as sensitized lymphocytes that have been immortalizedwith an oncogene may be used to obtain antibody.

A monoclonal antibody thus produced can also be obtained as arecombinant antibody by recombinant gene technology (see, for example,Borrebaeck, C. A. K., and Larrick, J. W., THERAPEUTIC MONOCLONALANTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERSLTD., 1990). Recombinant antibody may be produced by cloning DNAencoding it from the hybridoma or an immune cell such as sensitizedlymphocytes that produce antibodies, and integrating it into a suitablevector, which is then introduced into a host to produce said antibody.The present invention also encompasses such recombinant antibodies.

Antibodies of the present invention may be antibody fragments ormodified versions thereof as long as they bind the protein of thepresent invention. For example, as fragments of antibody, there may bementioned Fab, F(ab')2, Fv or single-chain Fv (scFv) in which Fv or Fv'sof the H chain and the L chain were ligated via a suitable linker(Huston, J. S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879–5883).Specifically, antibodies are treated with an enzyme such as papain orpepsin, to produce antibody fragments, or genes encoding these antibodyfragments are constructed and then introduced into an expression vector,which is then expressed in a suitable host cell (see, for example, Co,M. S. et al., J. Immunol. (1994) 152, 2968–2976; Better, M. and Horwitz,A. H., Methods in Enzymology (1989) 178, 476–496; Pluckthun, A. andSkerra, A., Methods in Enzymol. (1989) 178, 497–515; Lamoyi, E., Methodsin Enzymol. (1986) 121, 652–663; Rousseaux, J. et al., Methods inEnzymol. (1986) 121, 663–669; Bird, R. E. and Walker, B. W., TrendsBiotechnol. (1991) 9, 132–137).

As modified antibodies, antibodies bound to various molecules such aspolyethylene glycol (PEG) can also be used. The term “antibody” as usedherein encompasses these modified antibodies. In order to obtain thesemodified antibodies, the antibody obtained may be chemically modified.These methods are established in the field of the art.

Furthermore, the antibody of the present invention may be obtained as achimeric antibody comprising a variable region derived from a non-humanantibody and a constant region derived from a human antibody, or as ahumanized antibody comprising a complementarity determining region (CDR)derived from a non-human antibody and a framework region (FR) and aconstant region derived from a human antibody.

Antibodies obtained as described above can be purified to homogeneity.Separation and purification of the antibody for use in the presentinvention may be accomplished by the separation and the purificationmethods conventionally used for protein. For example, antibody can beseparated and purified by selecting and combining, but not limited to,chromatography columns such as affinity chromatography, filtration,ultrafiltration, salting-out, dialysis, SDS polyacrylamide gelelectrophoresis, and isoelectric focusing as appropriate (Antibodies: ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory, 1988). The measurement of concentration of the antibodyobtained as above can be determined by the measurement of absorbance,enzyme-linked immunosorbent assay (ELISA), and the like.

As columns for use in affinity chromatography, there can be mentioned aProtein A column and a Protein G column. Examples of the columns used inthe Protein A column are Hyper D, POROS, Sepharose F. F. (Pharmacia) andthe like.

As chromatography other than the above-mentioned affinitychromatography, there can be mentioned, for example, ion exchangechromatography, hydrophobic chromatography, gel-filtration, reversephase chromatography, adsorption chromatography, and the like(Strategies for Protein Purification and Characterization: A LaboratoryCourse Manual, Ed Daniel R. Marshak et al., Cold Spring HarborLaboratory Press, 1996). These chromatographies can be carried out usinga liquid chromatography such as HPLC and FPLC.

As the method of determining antibody binding activity of the antibodyof the present invention obtained as above, there can be usedenzyme-linked immunosorbent assay (ELISA), enzymeimmunoassay (EIA),radioimmunoassay (RIA), or fluorescent antibody assay. When ELISA isused, the protein of the present invention is added to a plateimmobilized with the antibody of the present invention, and then asample containing the antibody of interest, for example a culturesupernatant of the antibody-producing cells or purified antibody isadded.

The antigen binding activity can be evaluated by adding a secondantibody that recognizes the antibody labeled with an enzyme such asalkaline phosphatase, incubating and then washing the plate, andsubsequently, after adding the enzyme substrate such as p-nitrophenylphosphate thereto, determining absorbance. As the protein, a fragment ofthe protein, a fragment comprising the C-terminal thereof, or a fragmentcomprising the N-terminal thereof may be used. For the evaluation ofactivity of the antibody of the present invention, BIAcore (Pharmacia)can be used.

By using these techniques, the method of detecting or determining theprotein of the present invention can be performed, said methodcomprising bringing the antibody of the present invention into contactwith a sample expected to contain the protein of the present invention,and detecting or determining an immune complex of said antibody and saidprotein or immunostaining the cells that express said protein using saidantibody.

The method of detecting or determining the protein of the presentinvention can specifically detect or determine the protein and,therefore, is useful in various experiments using the protein.

The present invention also relates to DNA that specifically hybridizesto DNA (SEQ ID NO: 1) encoding the human WT1 interacting protein or DNAcomplementary to said DNA, and that has a chain length of at least 15bases. The term “specifically hybridize” means that there occurs nosignificant cross-hybridization with DNA encoding other proteins under astandard hybridization conditions, preferably under a stringenthybridization condition. Such DNA includes a probe, a primer, anucleotide or a nucleotide derivative (for example, antisenseoligonucleotide or ribozyme) that can specifically hybridizes to DNAencoding the protein of the present invention or DNA complementary tosaid DNA. Furthermore, such DNA can be used for the preparation of DNAchips.

The present invention includes, for example, an antisenseoligonucleotide that hybridizes to any site in the base sequence of SEQID NO: 1. The antisense oligonucleotide is preferably an antisenseoligonucleotide against at least 15 or more contiguous nucleotides inthe base sequence as set forth in SEQ ID NO: 1. More preferably, it isthe above antisense oligonucleotide in which the above contiguous atleast 15 or more nucleotides contain a translation initiation codon.

As the antisense oligonucleotide, their derivatives or modified versionsmay be used. As such modified versions, there can be mentioned, forexample, lower alkylphosphonate modified versions such as methylphosphonate or ethyl phosphonate type, phosphorothioate modifiedversions, or phosphoramidate modified versions.

As used herein “antisense oligonucleotide” may contain one or aplurality of nucleotide mismatches as long as nucleotides correspondingto nucleotides constituting a given region of DNA or mRNA are allcomplementary, and DNA or mRNA and the oligonucleotide can specificallyhybridize to the base sequence as set forth in SEQ ID NO: 1.

Such DNA is a region of at least 15 contiguous nucleotide sequence andhas a homology of at least 70%, at least 80%, and at least 90%, and morepreferably 95% or more on the base sequence. The algorithm used fordetermining homology may be one described herein. Such DNA is useful asa probe for detecting or isolating DNA encoding the protein of thepresent invention and as a primer for amplifying it as described below.

The antisense oligonucleotide derivative of the present invention bindsto DNA or mRNA encoding said protein by acting on the cells that producethe protein of the present invention, thereby inhibit its transcriptionor translation, or promote the decomposition of mRNA, resulting in thesuppression of expression of the protein of the present invention.Eventually it exhibits an effect of suppressing the actions of theprotein of the present invention.

The antisense oligonucleotide derivative of the present invention can bemixed with an appropriate base that is inert thereto to formulate anexternal preparation such as a liniment, a cataplasm and the like.

It can also be mixed, as desired, with an excipient, an isotonic agent,a solubilizer, a stabilizer, an antiseptic, a soothing agent or the liketo formulate a tablet, powders, granules, a capsule, a liposome capsule,an injection, a solution, a nasal drop, and the like as well as alyophilized preparation. They can be prepared according to conventionalmethods.

The antisense oligonucleotide derivative of the present invention may beapplied to the patient by either directly administering to the affectedarea of the patient or administering into the blood vessel therebyallowing the substance to be delivered to the affected area.Furthermore, an antisense encapsulating material that enhances prolongedaction and membrane permeability may be used. There may be mentioned,for example, liposome, poly-L-lysine, lipid, cholesterol, lipofectin orderivatives thereof.

Preferably the dosage of the antisense oligonucleotide derivative of thepresent invention can be adjusted and a preferred amount can be employedas appropriate depending on the condition of the patient. For example, apreferred dosage is in the range of 0.1 to 100 mg/kg, preferably 0.1 to50 mg/kg.

The antisense oligonucleotide of the present invention is useful ininhibiting the expression of the protein of the present invention, andthereby in suppressing the biological activity of the protein of thepresent invention. An inhibitor of expression containing the antisenseoligonucleotide of the present invention can suppress the biologicalactivity of the protein of the present invention, and therefore, isuseful.

The protein of the present invention is useful for screening compoundsthat bind thereto. Thus, it is used in a method of screening a compoundthat binds to the protein of the present invention, said methodcomprising bringing the protein of the present invention into contactwith a test sample expected to contain a compound that binds to saidprotein, and selecting a compound having an activity of binding to theprotein of the present invention.

The protein of the present invention for use in the screening may be arecombinant protein or a naturally occurring protein. Alternatively itmay be a partial peptide. Test samples include, but are not limited to,cell extracts, cell culture supernatants, microbial fermentationproducts, marine organism extracts, plant extracts, purified or roughlypurified proteins, peptides, non-peptide compounds, synthetic compounds,and naturally occurring compounds. The protein of the present inventionto be contacted with the test sample can be contacted with the testsample as a purified protein, as a form bound to a carrier, and as afusion protein with another protein.

As a method of screening a protein (ligand etc.) that binds to thepresent invention using the protein of the present invention, variousmethods known to a person skilled in the art can be used. Such screeningcan be performed by, for example, immunoprecipitation. Specifically, itcan be carried out as follows. A gene encoding the protein of thepresent invention is inserted into a vector for expressing foreign genessuch as pSV2neo, pcDNA I and pCD8 so as to permit the expression of saidgene in an animal cell.

As the promoter used in expression, any commonly used promoters may beused such as SV40 early promoter (Rigby In Williamson (ed.), GeneticEngineering, Vol. 3. Academic Press, London, pp. 83–141 (1982), EF-1 αpromoter (Kim et al., Gene 91, 217–223, 1990), CAG promoter (Niwa etal., Gene 108, 193–200, 1991), RSV LTR promoter (Cullen Methods inEnzymology 152, 684–704, 1987), SR α promoter (Takebe et al., Mol. Cell.Biol. 8, 466, 1988), CMV immediate early promoter (Seed and Aruffo,Proc. Natl. Acad. Sci. USA 84, 3365–3369, 1987), SV40 late promoter(Gheysen and Fiers, J. Mol. Appl. Genet. 1, 385–394, 1982), Adenoviruslate promoter (Kaufman et al., Mol. Cell. Biol. 9, 946, 1989), and HSVTK promoter.

In order to express a foreign gene by introducing the gene into ananimal cell, there are the electroporation method (Chu, G. et al.,Nucleic Acids Res. 15, 1311–1326, 1987), the calcium phosphate method(Chen, C and Okayama, H., Mol. Cell. Biol. 7, 2745–2752, 1987), the DEAEdextran method (Lopata, M. A. et al., Nucleic Acids Res. 112, 5707–5717,1984; Sussman, D. J. and Milman, G., Mol. Cell. Biol. 4, 1642–1643,1985), the lipofectin method (Derijard, B., Cell 7, 1025–1037, 1994;Lamb, B. T. et al., Nature Genetics 5, 22–30, 1993; Rabindran, S. K. etal., Science 259, 230–234, 1993), and the like, and any of them may beused.

By introducing a recognition site (epitope) of a monoclonal antibody, ofwhich the specificity is known, into the N-terminal or C-terminal of theprotein of the present invention, the protein of the present inventioncan be expressed as a fusion protein having the recognition site of themonoclonal antibody. As the epitope-antibody system, commerciallyavailable ones may be used (Jikken Igaku (Experimental Medicine), 13,85–90, 1995). Vectors are commercially available that permit theexpression of fusion proteins with β-galactosidase, maltose-bindingprotein, glutathione S-transferase, green fuorescence protein (GFP), andthe like, via multiple cloning sites.

A method of preparing a fusion protein has also been reported in which asmall epitope portion alone comprising a few to about a dozen aminoacids is introduced so that the effect of the resulting fusion proteinon changes in the property of the protein of the present invention isminimal. For example, an epitope such as poly-histidine (His-tag),influenza hemagglutinin (HA), human c-myc, FLAG, vesicular stomatitisvirus glycoprotein (VSV-GP), T7 gene10 protein (T7-tag), herpes simplexvirus glycoprotein (HSV-tag), E-tag (epitope on the monoclonal phage)and the like and the monoclonal antibody that recognizes it can be usedas the epitope-antibody system for screening proteins that bind to theprotein of the present invention (Jikken Igaku (Experimental Medicine)13, 85–90, 1995).

In immunoprecipitation, an immune complex is formed by adding theseantibodies to a cell lysate prepared by using a suitable surfactant. Theimmune complex comprises the protein of the present invention, a proteincapable of binding thereto, and an antibody. In addition to using anantibody to the above epitope, an antibody to the protein of the presentinvention can also be used to perform immunoprecipitation. The antibodyto the protein of the present invention can also be prepared byintroducing a gene encoding the protein of the present invention into asuitable E. coli expression vector to express said protein in E. coli,purifying the expressed protein, and then using this to immunize arabbit, a mouse, a rat, a goat, a chicken, and the like. It can also beprepared by immunizing the above animal with a partial peptide of theprotein of the present invention that was synthesized.

An immune complex can be precipitated by using, for example, Protein ASepharose or Protein G Sepharose when the antibody is a mouse IgGantibody. When the protein of the present invention was prepared as afusion protein with an epitope such as GST, an immune complex can beformed, as when the antibody of the protein of the present invention wasused, by using a substance that specifically binds to these epitopessuch as glutathione-Sepharose 4B.

For a general method of immunoprecipitation, it is carried out accordingto, or persuant to, the method described in an article (Harlow, E. andLane, D.: Antibodies, pp. 511–552, Cold Spring Harbor Laboratorypublications, New York, 1988).

For the analysis of immunoprecipitated protein, SDS-PAGE is generallyused, in which the bound protein can be analyzed based on the molecularweight of the protein by using a suitable concentration of gel. At thistime, generally the protein bound to the protein of the presentinvention cannot be detected by a standard staining method for proteinsuch as Coomassie staining or silver staining, and therefore the cellsare cultured in a culture liquid containing ³⁵S-methionine or³⁵S-cycteine to label the protein in the cell, which is then detected inorder to enhance the sensitivity of detection. If the molecular weightof the protein is known, the protein of interest can be directlypurified from the SDS-polyacrylamide gel electrophoresis, and thesequence can be determined.

As a method of isolating a protein bound to said protein using theprotein of the present invention, the west western blot (Skolnik, E. Y.et al., Cell (1991) 65, 83–90), for example, can be used. Thus, a cDNAlibrary is constructed using a phage vector (λgt11, ZAP etc.) from acell, a tissue, and an organ (for example, tissues, cells, and culturedcells such as the heart, the placenta, the testis, the thymus, andperipheral leukocytes) in which the binding protein that binds to theprotein of the present invention is expected to be expressed, and to thelibrary is expressed on the LB-agarose to immobilize the expressedprotein on a filter. Then the purified and labelled protein of thepresent invention and the above filter are reacted, and a plaque thatexpresses the protein bound to the protein of the present invention isdetected based on the label.

As methods of labelling the protein of the present invention, there canbe mentioned a method of utilizing the binding property of biotin andavidin, a method of utilizing an antibody that specifically binds to theprotein of the present invention or a peptide or a polypeptide (forexample, GST) fused to the protein of the present invention, a method ofutilizing radioisotope, a method of utilizing fluorescence, and thelike.

As another aspect of the screening method of the present invention,there can be mentioned a method of conducting a 2-hybrid system (Fields,S. and Sternglanz, R., Trends. Genet. (1994) 10, 286–292) that usescells. The protein of the present invention is fused to the SRF DNAbinding region or the. GAL4 DNA binding region, and then is expressed inyeast cells. From the cells expected to express the protein that bindsto the protein of the present invention, a cDNA library is constructedthat expresses, in a form fused with the VP16 or the GAL4 transcriptionactivation region, which is introduced into the above yeast cells. Fromthe positive clones detected, library-derived cDNA is isolated andintroduced into E. coli for expression (when the protein that binds tothe protein of the present invention is expressed in the yeast cells,the binding of the two results in the activation of the reporter gene,and thereby positive clones can be confirmed.).

It is also possible to prepare a protein that binds to the protein ofthe present invention or the gene thereof using the “two-hybrid system”(MATCHMARKER Two-Hybrid System”, “Mammalian MATCHMARKER Two-Hybrid AssayKit”, “MATCHMARKER One-Hybrid System” (all manufactured by Clontech),“HybriZAP Two-Hybrid Vector System” (manufactured by Stratagene), and anarticle “Dalton S. and Treisman R. (1992) Characterization of SAP-1, aprotein recruited by serum response factor to the c-fos serum responseelement. Cell 68, 597–612”). As the reporter gene, the Ade2 gene, theLacZ gene, the CAT gene, the luciferase gene, the PAI-1 (plasminogenactivator inhibitor type 1) gene, and the like can be used in additionto the HIS3 gene.

The screening of a compound that binds to the protein of the presentinvention can also be effected using affinity chromatography. Forexample, the protein of the present invention is immobilized onto anaffinity column, to which a test sample expected to be expressing aprotein that binds to the protein of the present invention is applied.As the test sample in this case, for example, cell extracts, celllysates and the like may be mentioned. After applying the test sample,the column is washed, and the protein that is bound to the protein ofthe present invention can be prepared.

The protein obtained is analyzed for its amino acid sequence and, basedon the sequence, an oligo DNA is synthesized. Using said DNA as theprobe, a DNA library can be screened so that DNA encoding said proteincan be obtained.

In accordance with the present invention, as a means to detect ordetermine the compound that is bound, a biosensor that utilizes thesurface plasmon phenomenon can be used. The biosensor that utilizes thesurface plasmon phenomenon permits realtime observation, as a surfaceplasmon resonance signal, of the interaction between the protein of thepresent invention and the test compound using a trace amount of proteinwithout labeling it (for example, BIAcore, manufactured by Pharmacia).Thus, by using a biosensor such as BIAcore, the binding of the proteinof the present invention and the test compound can be evaluated.

As methods of isolating compounds that bind to the protein (including anagonist and an antagonist) of the present invention, in addition toprotein, a method in which a synthetic compound, a natural product bank,or a random phage peptide display library is acted onto the immobilizedprotein of the present invention, and molecules that bind to the proteinof the present invention are screened, and a screening method using ahigh throughput using the combinatorial chemistry technology (Wrighton NC; Farrell F X; Chang R; Kashyap A K; Barbone F P; Mulcahy L S; JohnsonD L; Barret R W; Jolliffe L K; Dower W J., Small peptides as potentmimetics of the protein hormone erythropoietin, Science (United States)Jul. 26, 1996, 273 pp. 458–64, Verdine G L., The combinatorial chemistryof nature, Nature (England) Nov. 7, 1996, 384 pp. 11–13, Hogan J C Jr.,Directed combinatorial chemistry, Nature (England) Nov. 7, 1996, 384 pp.17–9) are known to a person skilled in the art.

Furthermore, the present invention relates to a method of screening acompound that promotes or inhibits the activity of protein of thepresent invention. As the WT1 interacting protein of the presentinvention has an activity of binding to the WT1 protein and therebyregulating the function of the WT1 protein, this activity can be used asan index to screen a compound that promotes or inhibits the activity ofthe WT1 interacting protein of the present invention.

The screening method comprises the steps of (a) culturing the cells thatexpress the WT1 interacting protein in the presence of a sample to betested, (b) detecting the growth of said cells, and (c) selecting acompound that promotes or inhibits said growth as compared to whendetected in the absence of a sample to be tested.

The protein used in screening is not specifically limited as long as ithas an activity of regulating the function of the WT1 protein. Forexample, the human WT1 interacting protein can be mentioned, and aprotein that is functionally equivalent to this protein can also so beused. Also, the WT1 interacting protein may be intracellularly orextracellularly expressed by the cell.

Test samples are not specifically limited, and there may be mentionedcell extracts, cell culture supernatants, microbial fermentationproducts, marine organism extracts, plant extracts, purified or roughlypurified proteins, peptides, non-peptide compounds, synthetic compounds,and naturally occurring compounds. It is also possible to use thecompound obtained in the screening of compounds that bind to the proteinof the present invention, as the test compound.

The compound isolated in this screening can be an agonist or anantagonist candidate for the protein of the present invention. The term“agonist” as used herein means a molecule that activates the function ofthe protein of the present invention by binding thereto. Also the term“antagonist” as used herein means a molecule that suppresses thefunction of the protein of the present invention by specifically bindingthereto. Furthermore, it can be a candidate compound that inhibits theinteraction of the molecule (including DNA and protein) that interactswith the protein of the present invention.

The detection of cell growth can be effected by, for example, detectingthe measurement of colony forming rate and the determination of thegrowth rate of the cell, the measurement of cell cycle, and the like.

The compounds isolated from these screenings can become candidate drugsfor promoting or inhibiting the activity of the protein of the presentinvention, and its application into the treatment of diseases (forexample cancer) with which the protein of the present invention isassociated, is conceivable.

Substances, obtained using the screening method of the presentinvention, in which part of the structure of compounds that promote orinhibit the activity of the WT1 interacting protein has been modified byaddition, deletion and/or substitution is also included in the compoundsobtained by the screening method of the present invention.

The present invention also relates to a method of screening a compoundthat promotes or inhibits the binding of a WT1 interacting protein ofthe present invention with the WT1 protein. The method comprises thesteps of: (a) allowing the WT1 interacting protein to react with the WT1protein in the presence of a sample to be tested, (b) determining thebinding activity of both proteins, and (c) selecting a compound thatpromotes or inhibits said binding as compared to when detected in theabsence of the sample to be tested.

As a method of assaying whether a compound contained in the test samplepromotes or inhibits the binding of the present protein and the WT1protein, there may be mentioned the west western blotting method or amethod of quantitating the conjugate of the both proteins in a solution(in vitro) or in the cell (in vivo).

When the compound obtained by the screening method of the presentinvention is used as a drug for humans and mammals such as mice, rats,guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cattle,monkeys, baboons, and chimpanzees, the isolated compound can also beformulated according to a known pharmaceutical method foradministration, in addition to directly administering the compounditself to patients.

For example, it can be used either orally as sugar-coated tablets,capsules, elixirs or microcapsules, or parenterally in the form ofaseptic solutions with water or another pharmaceutically acceptableliquid or injections of a suspension. For example, conceivably, it iscombined with a pharmaceutically acceptable carrier or a medium,specifically sterile water or physiological saline, a plant oil, anemulsifying agent, a suspension, a surfactant, a stabilizer, a flavoringagent, an excipient, a vehicle, a preservative, a binder and the like,and mixed in a unit dosage form, required for commonly recognizedpharmaceutical practice, to formulate into a drug. The amount of anactive ingredient in these formulations is adjusted to provide asuitable amount in the indicated range.

As additives that can be mixed into tablets or capsules, there can beused, for example, a binder such as gelatin, corn starch, gum tragacanthand gum Arabic, an excipient such as crystalline cellulose, a swellingagent such as corn starch, gelatin and alginic acid, a lubricant such asmagnesium stearate, a sweetening agent such as sucrose, lactose andsaccharin, and a flavoring agent such as peppermint, gaultheria oil orcherry. When the formulation dosage unit is a capsule, the abovematerials can further contain liquid carriers like oil and fat. Sterilecompositions for injection can be formulated according to a normalpharmaceutical practice using a vehicle such as distilled water forinjection.

As an aqueous solution for injection, there can be mentioned, forexample, physiological saline, an isotonic liquid containing glucose andanother adjuvant, for example, D-sorbitol, D-mannose, D-mannitol andsodium chloride, and may be combined with a suitable solubilizing agentsuch as alcohol, specifically ethanol, polyalcohol such as propyleneglycol, polyethylene glycol, non-ionic surfactant such as polysorbate 80(™TM), and HCO-50.

As an oleaginous solution, there may be mentioned sesame oil and soybean oil, and as a solubilizing solution, benzyl benzoate or benzylalcohol may be used in combination. Furthermore, a buffer such asphosphate buffer, sodium acetate buffer, a soothing agent such asprocaine chloride, a stabilizer such as benzyl alcohol and phenol, anantioxidant may be blended. The prepared injection is usually filledinto a suitable ampoule.

The administration to patients may be performed by intraarterialinjection, intravenous injection, subcutaneous injection, and nasally,transbronchially, intramuscularly, or orally by a method known to aperson skilled in the art. The dosage may vary depending on the weightand age of patients, method of administration and the like, and a personskilled in the art can select a suitable dosage as appropriate. Genetherapy is also contemplated in which said DNA is integrated into avector for gene therapy. The dosage method of administration may varydepending on the weight and age of patients, and the like, and a personskilled in the art can select a suitable dosage as appropriate.

For example, the dosage of a compound that binds to the protein of thepresent invention or a compound that inhibits the activity of theprotein of the present invention may vary depending on the diseasecondition, but in the case of oral administration, dosage for an adult(assuming body weight being 60 kg) is about 0.1 to 100 mg/day,preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg.

In the case of the parenteral administration, the unit dosage may varydepending on the subject to be administered, the subject organ, diseasecondition, and the method of administration, but in the form of aninjection, a convenient dosage for an adult (assuming body weight being60 kg) is about 0.01 to 30 mg/day, preferably about 0.1 to 20 mg, andmore preferably about 0.1 to 10 mg injected intravenously. For otheranimals, the amount converted from body weight of 60 kg may beadministered.

EXAMPLES

Now the present invention will be explained in more detail hereinbelow.

Example 1

Purification of WTIP

(1) Construction and Purification of GST Fusion Protein

Using pBluescript II/WT1(+/+) (Kudoh T. et al., Oncogene, Vol. 13, pp.1431–1439, 1996) as the template, PCR was conducted using the followingprimers:

1) GST-WT2 5′-TTGAATTCAATGGGCTCCGACGTGCGG-3′ (SEQ ID NO: 3)5′-TTGTCGACCATGGGATCCTCATGCTT-3′ (SEQ ID NO: 4) 2) GST-WT35′-TTGAATTCAATGGGCTCCGACGTGCGG-3′ (SEQ ID NO: 5)5′-TTGTCGACGAAGACACCGTGCGTGTG-3′ (SEQ ID NO: 6) 3) GST-WT45′-TTGAATTCAGATCCAATGGGCCAGCAC-3′ (SEQ ID NO: 7)5′-TTGTCGACGAAGACACCGTGCGTGTG-3′ (SEQ ID NO: 8)in which an EcoRI recognition sequence has been added to the 5′-end anda NotI recognition sequence has been added to the 3′-end to constructinsertion fragments. To the regions that are difficult to amplify,restriction enzyme fragments were inserted via a plasmid as appropriate.The base sequence of the amplified fragment is 1–882 for GST-WT3, 1–546for GST-WT2, and 541–882 for GST-WT4 (B in FIG. 1). The insertionfragment was integrated into pGEX-5X-3 (Amersham Pharmacia Biotech) andthen the sequence was confirmed, and transformed into an E. coli strainBL21 (DE3). At this time, pT-Trx, a thioredoxin expression vector, wascoexpressed in order to enhance the solubility of the fusion protein(Yasukawa T. et al., J. Biol. Chem. Vol. 270, pp. 25328–25331, 1995).

After confirming expression, an overnight culture was diluted 10-foldand incubated at 37° C. for 1.5 hour, to whichisopropyl-β-D-thiogalacto-pyranoside (IPTG) was added to a finalconcentration of 0.1 mM. After further culturing for 5 hours, the cellswere collected, to which a cell dissolution buffer (50 mM Tris-HCl, pH7.5, 50 mM NaCl, 1 mM EDTA, 1 mM Pefabloc SC (Boehringer Mannheim), 10μg/ml leupeptin, 1 mM DTT) was added, and solubilized by sonication. Thefusion protein in the supernatant was bound to the glutathione Sepharose4B (Amersham Pharmacia Biotech), and eluted with an elution buffer (50mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM reduced glutathione, 1 mM DTT).The protein concentration was determined by the Bradford method (ProteinAssay, Bio-Rad) and bovine serum albumin was used as a standard.

(2) Detection of WTIP by West Western Blot

In order to investigate the presence of the WT1 interacting protein thatcan be detected with the GST fusion protein in the K562 cells, K562(5×10⁶) was dissolved in 500 μl of the sample buffer for SDS-PAGE, andthe west western blot was carried out.

After separating the sample protein on SDS-PAGE, it was transferred to aPVDF membrane (Immobilon-P, Millipore) by the semi-dry method, blockedwith TBST (0.05% Tween 20) containing 2% skim milk, and was reacted with10–20 μg/ml TBS for 1 hour to overnight.

After washing the PVDF membrane with TBST, it was reacted for 1 hourwith a 1000-fold dilution of anti-GST antibody (Santa Cruz) in TBST,washed again in TBST, and then reacted for 1 hour with a 8000-folddilution of anti-mouse IgG antibody (alkaline phosphatase(ALP)-conjugated, or horseradish peroxidase (HRP)-conjugated) in TBST.After washing in TBST and TBS, it was subjected to color developmentwith a NBT/BCIP solution for the ALP-conjugated antibody, and wassubjected to light emission with ECL for the HRP-conjugated antibody.

FIG. 2 shows the result of the west western blot. A band at about 115kDa observed in GST-WT2 and GST-WT3 was not recognized in GST,indicating that this is a specific band showing the presence of the WT1interacting protein. Furthermore, since the same band was not recognizedin GST-WT4, it was shown to have a binding site in the 182 amino acidresidues at the N-terminal end of WT1. Since there had been no reportson the WT1 interacting protein of this size (p115), its separation andpurification were attempted.

(3) Purification of WTIP

Sample Preparation

After washing the K562 cells (6×10⁸ cells) (about 140 mg of protein) inPBS, it was dissolved in 27 ml of the cell dissolution buffer (50 mMTris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM Pefabloc, 10 μg/ml),disrupted by sonication at 60 W for 30 minutes, followed by the additionof 3 ml of the cell dissolution buffer containing 10% SB-12 and furthersonication twice at 60 w for 30 minutes, and the final concentration wasmade 150 mM in 5M NaCl. It was centrifuged to separate the supernatant,filtered with a 0.45 μm filter to prepare a sample for FPLC (AmershamPharmacia Biotech).

Chromatography

An anion exchange chromatography was performed using the HiLoad 16/10 QSepharose High Performance (Amersham Pharmacia Biotech) and eluted at aflow rate of 2 ml/min with a salt concentration gradient of 0 M to 0.5 MNaCl. The starting buffer was 20 mM Tris-HCl, pH 7.5, 0 M NaCl, 1 mMEDTA, 0.1% CHAPS, and the elution buffer was the starting buffer+0.5 MNaCl. From each of the eluted fractions 20 μl was aliquoted and wassubjected to a west western blot using GST-WT3, and the fraction (FIG.3) for which the WT1 interacting protein was detected was used for thesubsequent purification.

Using the same buffer, an anion exchange chromatography was performedusing MONO Q HR 5/5 (Amersham Pharmacia Biotech). The flow rate was 1ml/min. A similar assay was performed for each fraction (FIG. 4). Ahydrophobic interaction chromatography was performed using PhenylSuperose HR 5/5 (Amersham Pharmacia Biotech), and then was eluted at aflow rate of 0.5 ml/min and a salt concentration gradient of 1.0 M to 0M. The starting buffer was 20 mM Tris-HCl, pH 7.5, 1.0 M (NH₄)2SO₄, 1 mMEDTA, and the elution buffer was 20 mM Tris-HCl, pH 7.5, 0 M (NH₄)2SO₄,1 mM EDTA. Assay of each fraction was performed by west western blot asin the above, and from the fraction in which the WT1 interacting proteinwas detected (FIG. 5), protein was recovered with trichloroacetic acid,separated on SDS-PAGE (5.0% gel), transferred to a PVDF membrane(proBlott, Applied Biosystem), and was used for peptide mapping.

Thus, as after hydrophobic interaction chromatography, a specific bandwas separated at around 115 kDa on SDS-PAGE, this was transferred to aPVDF membrane, subjected to CBB staining and west western blot toidentify a CBB band (FIG. 6). The bands was excised from the PVDFmembrane, and was directly used for peptide mapping.

Peptide Mapping

The protein transferred to the PVDF membrane was decomposed with lysilendopeptidase on the membrane, and after the formed peptide wasseparated on HPLC, the molecular weight of each peptide was determinedby a mass spectrometer (MALDI-TOF MS). The molecular weight pattern ofpeptides was compared to the amino acid sequence database to predict theoriginal protein.

Identification of WTIP

As a result of peptide mapping, the presence of three different proteinswas predicted in the band: HYPA/FGP11 (accession No. AF049528), RNAhelicase associated protein (AF083255), and siah binding protein 1(U51586). The base sequence at the 3′-end has not been determined forHYPA/FBP11, the base sequence at the 5′–end has not been determined forsia binding protein, and the entire sequence of the RNA helicaseassociated protein has been determined, but the molecular weight was77.9 kDa which was significantly different from that predicted based onthe position of the band. In order to identify the WT1 interactingprotein from among them, a his-tagged protein of the known region ofeach gene was constructed and its binding to GST-WT3 was investigated.As a result, since HYPA/FBP11 bound to GST-WT3, the cloning of thefull-length and the identification of the binding site were attempted.

The construction of the above his-tagged protein was performed asfollows:

Using cDNA of human leukemia cell line K562 or KG-1 as the template, andthe initiation codon of a primer (HYPA/FBP11 (AF049523)) to which a XhoIrecognition sequence was added at the 3′-end as 1, PCR was performed for1–1218 (amino acids 1–406), 1–534 (amino acids 1–178), 1–294 (aminoacids 1–98) bases to construct insertion fragments (B in FIG. 7), whichwere inserted into pET-21b(+) (Novagen) that adds a his tag to theC-terminal. In almost the same manner as for the GST fusion protein, E.coli was cultured, and the cells were recovered. However, pT-Trx was notused. The cells derived from 1 ml of the culture were dissolved in 100μl of the sample buffer for SDS-PAGE, and were directly used for westwestern blot and Western blot. The antibody used in Western blot wasanti-His tag antibody (c-term, Invitrogen).

Example 2

Cloning of cDNA Encoding WTIP

WTIP has a full length of about 3 kbp, and about 1.3 kbp at the 5′-endis HYPA/FBP11. This contains the WW domain that is known to bind to aproline-rich domain. Since the full-length of a mouse homolog of FBP11was reported, CDNA derived from K562 and from KG-1 was amplified usingthe sequence at the 3′-end, and an about 3 kbp fragment was obtained.

Using the CDNA of K562, PCR and RACE (RApid Amplification of cDNA ends)were performed from the known sequences. First, for the 5′-end, 5′ RACEwas performed using a base sequence 5′-CTTTGCTGGTTGGCTCTCCTCCTCTTCT-3′(SEQ ID NO: 9) in a known region as the antisense primer to confirm the5′-end of the known sequence.

For the 3′-end, using a base sequence5′-TTGATCATCATCCAGTTGCTCCAAAAGGG-3′ (SEQ ID NO: 10) corresponding to theC-terminal of the mouse FBP11 translation region as the antisense primerand a base sequence 5′-TGGGACAAATGCCTGGAATGATGTCGTC-3′ (SEQ ID NO: 11)in HYPA as the sense primer, PCR was performed to determine the basesequence, and it was found that this region contained NY-REN-6.

Furthermore, using a base sequence 5′-CAGCGATCAGAGTCTCGTTCTGCTTCAG-3′(SEQ ID NO: 12) in the NY-REN-6 region as the sense primer, 3′ RACE wasperformed to determine the base sequence. By combining these fragments,the full-length of WTIP was cloned.

An about 3 kbp DNA fragment obtained as above encodes the full-length ofhuman WTIP, and the base sequence is shown in SEQ ID NO: 1 and thededuced amino acid sequence in SEQ ID NO: 2. The determination of thebase sequence was performed by the TA cloning (Invitrogen) of cDNA. ForDNA database search, Basic BLAST (BLAST 2.0) on the NCBI server wasused. For the analysis of the base sequence, GENETYX-Mac 10.1 (SDCSoftware Development Institute) was used.

The structure of full-length WITP is shown in FIG. 7.

Example 3

WT1 Interaction Site for WTIP

The WW domain is known to bind to a prolin-rich domain having thePro-ProLeu-Pro (PPLP) (SEQ ID NO: 15) motif, but the same motif cannotbe found in Huntingtin. Though WT1 also has the proline-rich domain, itis not a PPLP (SEQ ID NO: 15) motif. In order to investigate whether WT1is bound to WTIP via the WW domain, west western blot was performedusing a his-tagged protein of a WTIP mutant. As shown in FIG. 8, amutant containing no WW domain had lost the ability of binding withGST-WT3, indicating that the WW domain is required for the binding ofWTIP and WT1.

Example 4

Reactivity of WT1 and WTIP in the Cell

Using a cell line U937 in which WT1 was forcefully expressed, aco-precipitation experiment by immunoprecipitation was performed toconfirm the binding of WT1 and WTIP in the cell.

1×10⁷ cells were dissolved in 500 μl of the ELB buffer (50 mM HEPES, pH7.5, 250 mM NaCl, 0.5 mM EDTA, 0.1% NP-40, 1 mM Pefabloc (™TM), Complete(™TM) EDTA free, 1 mM DTT), disrupted by sonication, allowed to stand onice for 1 hour, and centrifuged at 15000 rpm, 4° C. to recover thesupernatant. To the supernatant was added 4 μg of anti-WT1 antibody(C-19, Santa-Cruz), gently stirred at 4° C. for 2 hours, and then 20 μl(bed volume) of Protein A Sepharose (Amersham Pharmacia Biotech) wasadded, and was further stirred for 2 hours. After washing the Protein ASepharose 3 times with 1 mL of the ELB buffer, 40 μl of the samplebuffer for SDS-PAGE was added. The reaction supernatant was recovered asthe flow through, to which the sample buffer was similarly added. Thetwo were heated to 90° C. for 5 minutes, and was subjected to Westernblot. After electrophoresis on a 7.5% polyacrylamide gel, it wastransferred to Immobilon P (Millipore), and was subjected to colordevelopment with NBT/BCIP using anti-WTIP antibody as the primaryantibody and anti-rabbit IgG antibody (alkaline phosphatase-conjugated,Promega) as the second antibody.

In FIG. 9, FT represents flow through and IP represents the bindingfraction of Protein A Sepharose. At the position of the arrowhead inFIG. 9, a band of WTIP was observed. Thus, it was shown that WT1 andWTIP bind in the cells in which WT1 was forcefully expressed.

The invention claimed is:
 1. An isolated WT1 interacting proteincomprising the amino acid sequence as set forth in SEQ ID NO:
 2. 2. Anisolated protein that is encoded by DNA comprising the nucleotidesequence as set forth in SEQ ID NO:
 1. 3. An isolated function modulatorof the WT1 protein comprising as an active ingredient a polypeptideconsisting of the amino acid sequence as set forth in SEQ ID NO:
 2. 4. Amethod of producing a protein comprising culturing an isolated host cellcomprising a vector that comprising a sequence encoding the proteinaccording to claim
 1. 5. A method of screening a compound that binds toa protein according to claim or a partial peptide thereof 1, said methodcomprising: (a) bringing a sample to be tested into contact with saidprotein or said partial peptide thereof; (b) detecting the bindingactivity of said sample with said protein of said partial peptidethereof; and (c) selecting a compound comprising an activity of bindingto said protein or said partial peptide thereof.